The ability to respond to stimuli is often considered to be a key characteristic of life. Cells can detect new conditions, transduce that information into a usable form, and execute an appropriate response. One common signal transduction strategy is to represent information by the specific and transient placement of phosphoryl groups on proteins. Errors in signal transduction can lead to diseases (e.g. cancer, diabetes), and drugs have been developed to block aberrant signaling processes. Understanding the mechanisms, regulation, and impact of protein phosphorylation is thus of fundamental interest, as well as of practical significance to human health. Microorganisms are the dominant form of life on Earth by many measures, including genetic diversity, raw numbers, environmental distribution, and evolutionary experience. Thus, it is logical to seek basic signal transduction principles in microbes. Our long-term goal is comprehensive understanding of signal transduction by two-component regulatory systems, which occur in microorganisms from all three phylogenetic domains. In a typical two-component system, a sensor kinase detects stimuli and autophosphorylates. A response regulator then catalyzes phosphorylation from the sensor kinase (or from small molecules), which turns on the response. Response regulator dephosphorylation, either self-catalyzed or mediated by a phosphatase, ends the response. The kinetics of phosphoryl group reactions are important to synchronize responses with stimuli. Genome sequencing presents a challenge (a rapidly widening gap between the number of known proteins and what can be studied) and an opportunity (diverse and extensive sequence data). To learn how to reveal properties of tens of thousands of two-component proteins from sequence data alone, our innovative research strategy focuses on sequence differences (rather than similarities) between the conserved domains of sensor kinases or response regulators. We were productive during the previous grant period with an approach that integrated biochemistry, bioinformatics, biophysics, genetics, molecular biology, and structural biology. We identified factors that greatly affect response regulator reaction rates, but do not account for the entire known range. Our elucidation of the CheX mechanism, together with our previous work on CheZ, set the stage for a unified hypothesis of response regulator phosphatases. Building on our success, we will identify factors that affect phosphodonor binding and autophosphorylation (Aim 1), autodephosphorylation (Aim 2), and sensor kinase-mediated dephosphorylation (Aim 3) of response regulators and characterize underlying mechanisms. Antibiotic resistance of bacterial and fungal pathogens is a major and increasing threat to human health. Our study of the binding of small molecules to response regulators may influence design of therapeutic agents to disable critical two-component systems of microbial pathogens. The results of our project could also be used to predict or manipulate the signaling kinetics of two-component systems, or engineer synthetic regulatory circuits with specific timing characteristics. Fundamental principles of signal transduction may also emerge.